The present invention utilizes the major late promoter (MLP) from human adenovirus type 2. The MLP contains a unique start site (cap site) for the initiation of transcription, which is directed by two regulatory elements, the TATA region at approximately -25 to -31 relative to the cap site and a binding site for the major late transcription factor at approximately -52 to -64.
The present invention further utilizes an enhancer sequence to increase expression from the major late promoter of adenovirus. Many enhancer sequences are known and their ability to enhance gene expression have been well described in the literature. The preferred enhancer in this invention is from the human polyomavirus BK, strain P2 (Berg et al., 1988, Nucl. Acids Res. 16:9057). Although this enhancer stimulates expression from the late promoter in many cell lines, it is most useful in cells expressing the immediate-early gene products of a large DNA virus, such as the Ela gene product of adenoviruses. Under these conditions, the Ela protein unexpectedly stimulates BKV enhancer activity (Grinnell et al., 1988, Mol. Cell. Biol. 8:3448).
Also important to the present invention is the copolymer poly(dG-dT).poly(dA-dC), "the GT element". This element is widely dispersed in the eukaryotic genome, and has been found in the non-translated regions and introns of a number of known genes. The element is capable of forming left-handed DNA and has been suggested to play a role in recombination and gene conversion events. While this element has been suggested to have weak enhancer activity with the SV40 early promoter, it does not have enhancer activity when used in conjunction with the MLP (Berg et al., 1989, Mol. Cell. Biol. 9:5248). Quite unexpectedly, an enhancer activity can be elicited from the GT element in the presence of an immediate-early gene product of a large DNA virus, such as the Ela protein of adenovirus.
Another element pertinent to the invention is a synthetically-derived DNA sequence representing the tripartite leader (TPL) sequence from human adenovirus type 2. This sequence is found on late viral mRNAs and is believed to facilitate the translation of such viral messages at late times in the virus + infectious cycle, but not during early times following infection (Zhang et al., 1989, J. Biol. Chem. 264:10679 and Dolph et al., 1988, J. Virol. 62:2059). However, it has also been reported that efficient transcription, not translation, is dependent on adenovirus TPL sequences at late times post-infection (Alonso-Caplen, 1988, J. Virol. 62:1606), and that the enhancement of transcription required the entire first leader (to nt41), in addition to 149 nucleotides of the adjacent intron. It has been reported that the TPL stabilized non-adenovirus mRNAs only within the environment of an adenovirus-infected cell during the late phase of the infectious cycle (Moore and Shenk, 1988, Nucl. Acids Res. 16:2247). Thus from the published literature, the TPL would not be expected to dramatically enhance gene expression except at late times following viral infection. The synthetic sequence of the present invention is not utilized in adenovirus-infected cells under late in fection conditions. In contrast, the vectors are used in transformed cells expressing early viral functions. Further, the synthetic TPL does not contain the reported intron sequences required for transcriptional stimulation. Unexpectedly, the synthetic TPL of the present invention not only stimulates expression from the vectors of the present invention, but is approximately 2 fold more efficient than the previously disclosed nonspliced TPL sequence which does contain the sequence required for optimal transcription in late viral infected cells.